(a) Field of the Invention
The present invention relates to a genotyping kit and method for diagnosis of human papillomavirus (HPV) infection, probes for genotyping the HPV, and DNA chips including the probes. More specifically, the present invention relates to a genotyping kit for detecting human papillomaviruses from clinical samples of infected patients using a DNA chip, a process for preparing the said DNA chip, and a method for diagnosis of HPV infection using the genotyping kit.
(b) Description of the Related Art
Uterine cancer includes cervical cancer, endometrial cancer, uterine sarcoma and the like. For cervical cancer, approximately 450,000 new cases occur worldwide each year, approximately 6,000 of these in Korea. Since the occurrence of cervical cancer (including cervical intraepithelial neoplasia) accounts for 22.1% of total cancer cases in Korean women, the highest incidence with the second highest death rate, the prevention, diagnosis and treatment of cervical cancer are regarded as the most important issues in women's health.
Cervical cancer progresses through a precancerous stage, cervical intraepithelial neoplasia (CIN), known to be mainly caused by human papillomavirus (HPV) infection. Especially, infection by particular types of HPV raises the possibility of developing invasive disease. Over 70 genotypes of HPV have been identified since the recognition of HPV as the main etiological factor for cervical cancer, and through research, certain HPV genotypes have been selectively found in lesions of specific locations or progression stages, which has furthered recognition of the biological diversity of HPV infection. Among the. HPV genotypes detected in the anogenital area, over 10 genotypes have been classified as being part of the high-risk group that are associated with an elevated risk for developing cervical cancer. Based on these findings, characterization of the biological differences of HPV infection is considered to be of significant importance to the diagnosis and prevention of cervical cancer.
The test that has been most commonly used for the diagnosis of cervical cancer at its early stage is the Pap smear, which is a cytological test performed as follows: old cells removed from the outermost layer of cells from the surface of the cervix are stained and examined for histopathological characteristics of HPV infection including koilocytosis, perinuclear halo formation in the epithelial cells. However, due to the low diagnostic efficiency (1-15%) of Pap tests together with other limitations, additional methods such as colposcopy are necessary for more dependable diagnoses. Colposcopic screening can detect HPV infection at up to 70%, but it has disadvantages including high cost of the equipment, the need for skilled interpreters, and inability of determining HPV genotypes to distinguish between high-risk and low-risk infection. Therefore, efforts have been continuously made to develop techniques for the detection of HPV and identification of HPV genotypes, to supplement conventional screening methods for cervical cancer and its precursors such as the Pap test.
The methods for detection of HPV and identification of HPV genotypes can be classified into two groups, i.e., direct detection of HPV DNA and detection of amplified HPV DNA. The methods for direct detection of HPV DNA include liquid hybridization (Hybrid Capture kit by Digene Diagnostics, Silver Spring, Md., USA, www.digene.com), Southern blot and dot blot with HPV type-specific probes, filter in situ hybridization (FISH) and the like; and methods for the detection of amplified DNA include type-specific PCR (polymerase chain reaction) and general-primer PCR. In particular, genotype analyses of amplified HPV DNA by general primer sets are commonly performed by employing dot blot hybridization, microtitre plate hybridization, or a line probe assay. Among these methods, liquid hybridization by Hybrid Capture and line probe assay following a general-primer PCR have been considered most suitable for diagnostic purposes. The line probe assay can detect about 20 different HPV genotypes by immobilizing oligonucleotide probes on a nitrocellulose membrane, however it lacks reliability due to low sensitivity and difficulties in data interpretation. Commercialized Hybrid Capture kits can detect HPV DNA in clinical samples without PCR amplification, and they can distinguish between high-risk and low-risk HPV groups. But the fact that Hybrid Capture kits cannot identify the genotypes of infecting HPV limits accurate risk determination since the risk factors amongst high-risk HPVs are not the same, in other words, intermediate-risk types are included in the high-risk group. Moreover, the use of an RNA probe may present low stability of the kit, and the possibility of contamination cannot be excluded.
Under these circumstances, there have been strong reasons for exploring and developing a simple and accurate method and a genotyping kit for detection of HPV infection and identification of the genotype of infecting HPV with high specificity and sensitivity, as well as probes for genotyping the HPV, and DNA chips including the probes. In addition, the genotyping kit, the probes and the DNA chips must have specificity and sensitivity high enough to detect various kinds of HPV so that they can diagnose the HPV infection and identify its genotype with accuracy.